Overall Summary The overall goal of this proposal for renewal of P01 CA163222 is to further improve responses of metastatic melanoma to MAP kinase pathway-targeted and immune checkpoint therapies, by targeting aberrant tumor- intrinsic epigenetic regulation of gene expression, with a focus on enhancing the tumor immune microenvironment. While those revolutionary therapies have provided major improvements in melanoma outcomes, and advances in the past five years by us (in this Program Project) and others have substantially improved the outlook for overcoming therapeutic resistance, the majority of patients still experience eventual disease progression and fatality. Even with our significant successes in identifying resistance mechanisms and novel potential therapeutic strategies, there remains an urgent need to discover additional mechanisms and potential targets in acquired and intrinsic resistance to targeted and immune therapies for melanoma. Our current approach incorporates mechanistic insights uncovered by investigators under this Program Project Grant (PPG) and by other groups, namely the importance of genetic aberrations in epigenetic regulatory machinery in resistance to therapy, at least partially by reshaping the tumor immune microenvironment, and as potential targets for overcoming resistance. The three research projects in this proposal describe partially uncovered mechanisms by which different epigenetic and transcriptional regulators drive melanoma resistance to therapy and might be targeted therapeutically. These regulators include genomic copy number gains and newly described gain-of-function mutations in the histone methyltransferase G9a, relocalization in chromatin of the ETS transcription factor family member ETV1, and loss-of-function mutations in PBAF components of the SWI/SNF chromatin remodeling complexes. These regulators will be investigated in vitro and in multiple preclinical models in order to gain deeper understandings of their resistance mechanisms, particularly their effects on the anti-tumor immune response, as well as to identify biomarkers of response to and assess the feasibility of therapeutic strategies targeting them. Identification of resistance mediators and biomarkers of response will be corroborated in human pre- and on-treatment melanoma biopsies, which are readily available through our robust, independently funded Melanoma Patient Biopsy Program developed, in part, with funding from this P01. Complex integration and analysis of complementary epigenetic and transcriptional data (RNA- seq, ATAC-seq, and ChIP-seq) generated in all three projects will be performed by a Shared Resources Core using state-of-the-art computational analysis and validation models. The expected outcome of these studies is a more complete understanding of the mechanisms through which epigenetic and transcriptional regulators drive resistance to targeted and immune cancer therapies, setting the stage for development of clinical trials aimed at providing complete and durable responses to therapies for advanced melanoma and other cancers.